Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle that alternates between various species of small mammals and a tick vector. Like many bacterial pathogens, B. burgdorferi must adapt to a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual segmented genome that includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded genes are critical for successful adaptation by B. burgdorferi to the different environments that the spirochete encounters during its infectious cycle. A major focus of this project is to determine how and why the Lyme disease spirochete maintains such a unique genomic structure and the specific contributions of individual plasmids and genes at each stage of the infectious cycle. As described above, B. burgdorferi has multiple plasmids that are faithfully maintained as the bacterium grows and divides. Stable maintenance of low copy number plasmids requires replication and active partitioning to daughter cells. Both processes typically depend on plasmid-encoded products recognizing specific sites on the plasmid. On the basis of their sequence conservation, several Borrelia gene products have been implicated in plasmid replication and partition, and we previously demonstrated that shuttle vectors carrying these regions are capable of maintenance in B. burgdorferi. Analyzing the requirements for plasmid replication and partition in B. burgdorferi is complicated, however, by the complexity of the genome and the possibility that plasmid-encoded gene products may act in trans. In 2012, to avoid the complications presented by multiple co-existing replicons in B. burgdorferi, Kit Tilly and colleagues studied the replication/partition region of a B. burgdorferi plasmid in isolation, by inserting portions of it into an E. coli plasmid defective in partitioning, and subsequently analyzing the stability of the chimaeric plasmid in E. coli (Tilly, Checroun and Rosa, 2012, Plasmid, vol 68, pp1-12). These studies demonstrated that three genes from this B. burgdorferi plasmid, bbb10, bbb11 & bbb13, are required for stable plasmid maintenance, whereas one gene in this region, bbb12, is dispensable. This result was surprising because bbb12 is homologous to the essential partition protein ParA and the only gene in this region with a homolog in other bacteria. Another surprising aspect of these findings is that all three of the other genes in the region conferring autonomous replication in B. burgdorferi are required for plasmid stabilization in E. coli. Perhaps the BBB10, -11 and-13 products form a multi-protein complex that carries out both replication and partition functions. To validate the results obtained with this heterologous system, Kit attempted to inactivate two of these genes, bbb10 and bbb12, in B. burgdorferi. She obtained bbb12 mutants at a typical frequency, suggesting that this gene is not required for plasmid maintenance in B. burgdorferi or E. coli. Conversely, she was unable to inactivate bbb10 in B. burgdorferi. Together these results suggest that bbb10, bbb11 and bbb13 play crucial roles in plasmid maintenance, whereas bbb12 may encode an accessory factor. This study lays the foundation for more detailed biochemical studies of the plasmid-stabilizing functions of these B. burgdorferi gene products and demonstrates that their roles in plasmid replication and partitioning can be investigated in a less complex system. Several B. burgdorferi plasmids have been shown to encode genes that are critical for spirochete viability during its infectious cycle, where it must adapt to two very different environments, the Ixodes tick vector and a mammalian host. Linear plasmid lp54 is one of the most highly conserved plasmids in the B. burgdorferi genome and it also has one of the highest proportions of differentially regulated genes. Several genes on lp54 have been shown to contribute to spirochete fitness in vivo, but the majority of the proteins encoded on this plasmid are of unknown function and lack homologs in other organisms. We previously reported in FY2010 that deletion of a 4kb region of lp54 (bba01-bba07) led to a slight attenuation of this B. burgdorferi mutant (deltaA1-7) for tick-transmitted infection in mice by challenge with approximately 20 infected ticks. In FY2012, Aaron Bestor and colleagues reduced the tick challenge to a few ticks per mouse to create a more biologically relevant model (Bestor, Rego, Tilly and Rosa, 2012, Infection and Immunity, in press). Aaron found that this deltaA1-7 lp54 mutant was profoundly attenuated in its ability to infect mice by tick bite, whereas wild-type B. burgdorferi could routinely establish infection at a challenge dose as low as one tick per mouse. This finding was interesting and rather surprising because infectious dose studies demonstrated that the deltaA1-7 lp54 deletion mutant was as fully competent as wildtype spirochetes to infect mice by needle inoculation with either in vitro-grown or tick-derived spirochetes. Aaron next targeted the bba03 gene as the most likely candidate within the bba01-bba07 region of lp54 to contribute to spirochete transmission by tick bite. Previous studies have shown that BBA03, a putative outer membrane protein, is synthesized by B. burgdorferi during in vitro conditions that simulate tick feeding. Consistent with this hypothesis, Aaron demonstrated increased expression of bba03 and surface localization of the protein by spirochetes in fed relative to unfed ticks. However, inactivation of bba03 and assessment of the deltaA03 mutant throughout the infectious cycle indicated that BBA03 is not required by B. burgdorferi to establish mammalian infection by tick transmission. Given that BBA03 is a surface-exposed protein that is made specifically at the point of tick transmission, it was surprising to find that it is dispensable for completion of the experimental infectious cycle. To further probe the role of BBA03, Aaron co-infected ticks with wildtype and deltaA03 mutant spirochetes in order to more closely mimic the environment that B. burgdorferi typically encounters in the natural infectious cycle. In this context, the deltaA03 mutant demonstrated a significant defect in transmission and mouse infection, suggesting that BBA03 provides a competitive advantage to spirochetes carrying this gene during tick transmission to a mammalian host in the natural infectious cycle, where mixed infections with multiple B. burgdorferi strains are common. The discrete but critical point and circumstance during which BBA03 conveys a selectable advantage perhaps underlies the linkage on plasmid lp54 with other genes that play key roles during tick colonization and transmission. In FY2012 we collaborated with our extramural colleagues Dr. Jenifer Coburn at the Medical College of Wisconsin and Dr. Mollie Jewett at the University of Central Florida. Laura Ristow, a graduate student in Jenifer's lab, examined the role during infection of P66, an integral outer membrane protein of B. burgdorferi with adhesin and channel-forming activities. She demonstrated that P66 is essential for mammalian but not tick infection by B. burgdorferi (Ristow et al. 2012, Molecular Microbiology, in press). Sunny Jain, a graduate student in Mollie's lab, investigated the function and role of two plasmid genes in B. burgdorferi, which he showed encode purine permeases that are critical for mammalian infectivity (Jain et al. 2012, Infection and Immunity, 80:2831-40, 2012). Our contributions to both of these studies were discrete but significant at their inception.